The Microsporidia are emerging zoonotic pathogenic protists which are classified as category B priorty pathogens by the NIH and are also on the EPA high priority organism and contaminant lists. New drugs are needed, both for topical treatment of ocular microsporidiosis as well as systemic treatment of disseminated and gastrointestinal infections. This application represents an approach to rational drug design for these difficult pathogens. Our data, and that of other groups, indicates that fumagillin functions by inhibiting microsporidian methionine aminopeptidase type 2 (MetAP2) which is an essential enzyme in these organisms. We have cloned, expressed and determined the crystal structure of Encephalitozoon cuniculi MetAP2 (EcMetAP2) as well as developed yeast dependent on EcMetAP2 for growth. In addition, we have identified and cloned Enterocytozoon bieneusi MetAP2 (EbMetAP2). MetAP2 is a logical therapeutic target as microsporidia lack MetAP1 making MetAP2 an essential enzyme. Among eukaryotes this makes them highly susceptible to MetAP2 inhibitors and limits the toxicity of these compounds in their hosts as most eukaryotes have both MetAP1 and MetAP2. Use of fumagillin and its derivatives has confirmed that inhibition of MetAP2 is an effective in vitro and in vivo therapeutic target for many species of microsporidia suggesting that like E. cuniculi other microsporidia are dependent on MetAP2. Fumagillin has demonstrated efficacy in human infections due to Ent. bieneusi, but its use has been limited by bone marrow toxicity. Exploiting differences in the structure of MetAP2 between host and pathogen should permit the design of selective therapeutic competitive agents with decreased host toxicity. We plan to develop new competivie inhibitors of MetAP2 with increased selectivity for microsporidia, based on our EcMetAP2 crystal structure and in silico model of EbMetAP2. These inhibitors will be tested in vitro and in vivo for efficacy and in an iterative process we will use this information to refine our models and improve inhibitor design. We have assembled an integrated team with complementary expertise to develop and test these compounds. In addition, the target enzyme MetAP2 is important in other protozoa and our lead compounds should help define new classes of drugs that could have broad anti-parasitic activity and prove useful in the treatment of malaria and leishmaniasis.